Throughout this application various publications are referenced, many in parenthesis. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Deoxycytidylate deaminase (dCMP deaminase) catalyzes the deamination of dCMP to dUMP. This reaction provides the nucleotide substrate (dUMP) for thymidylate synthase (Jackson, R. C., J Biol Chem 253: 7440-7446 [1978]) and Chiu et al. (Chiu, C.-S. et al., J Biol Chem 252: 8603-8608 [1977]). The activity of the enzyme is controlled by the ratio of dCTP to dTTP in the cell. In this regard, dCMP deaminase is allosterically activated by dCTP and inhibited by dTTP (Maley, F. and Maley, G. F., Curr Topics Cell Regul 5: 177-228 [1972]). Evidence in support of this thesis was obtained by Xu and Plunkett using an in situ assay (Xu, Y.-Z. and Plunkett, W., Biochem Pharmacol 44: 1819-1827 [1992]). A consequence of this nucleotide interplay is that the amount of dUMP available for thymidylate synthase is finely controlled by the end products of the pyrimidine deoxynucleotide pathway.
Several observations indicate the importance of dCMP deaminase in DNA replication. First, deaminase activity has been shown to be elevated in such rapidly dividing tissue as regenerating liver (Maley, F. and Maley, G. F., J Biol Chem 235: 2968-2970 [1960]), chick and rat embryo (Maley, G. F. and Maley, F., J Biol Chem 234: 2975-2980 [1959]), and rat hepatomas (Maley, F. and Maley, G. F., Cancer Res 21: 1421-1426 [1961]). Second, HeLa cell dCMP deaminase activity is highest in late S phase and subsequently declines in the following G2 phase (Gelbard, A. S. et al., Biochim Biophys Acta 182: 564-566 [1969]). Finally, the absence of dCMP deaminase activity in mammalian cells induces an imbalance in deoxyribonucleotide pools such that dTTP levels decrease while dCTP levels increase (Bianchi, V. et al., Mol Cell Biol 7: 4218-4224 [1987]; de Saint Vincent, B. R. et al., J Biol Chem 255: 162-167 [1980]). This imbalanced pool can lead to increased mutation rates (Weinberg, G. et al., Proc Natl Acad Sci USA 78: 2447-2451 [1981]; Sargent, R. G. and Mathews, G. K., J Biol Chem 262: 5546-5553 [1987]; Meuth, M., Exp Cell Res 181: 305-316 [1989]).
dCMP deaminase has been purified to homogeneity from a variety of sources including human spleen (Ellims, P. H. et al., J Biol Chem 256: 6335-6340 [1981]); chick embryo (Maley, G. F. and Maley, F., in Advances in Enzyme Regulation, Weber, G., Ed., Vol. 8, pp. 55-71, Pergamon Press, London [1970]); donkey spleen (Geraci, G. et al., Biochemistry 6: 183-191 [1967]); T2r.sup.+ bacteriophage-infected Escherichia coli (Maley, G. F. et al., J Biol Chem 247: 931-939 [1972]); and HeLa cells (Maley, G. F. et al., Biochim Biophys Acta 1162: 161-170 [1992]). A dCMP deaminase gene from T4 bacteriophage was cloned and sequenced (Maley, G. F. et al., J Biol Chem 265: 47-51 [1990]) and compared with the complete amino acid sequence of the enzyme isolated from T2 bacteriophage-infected E. coli (Maley, G. F. et al., J Biol Chem 258: 8290-8297 [1983]). A homology comparison of the latter with a deduced amino acid sequence of dCMP deaminase from Saccharomyces cerevisiae has been made (Mcintosh, E. M. and Haynes, R. H., Mol Cell Biol 6: 1711-1721 [1986]), and several regions of similarity were found.
The cDNA for human dCMP deaminase has been cloned and expressed as a functional protein in Escherichia coli (Weiner, K. X. B. et al., J Biol Chem 268: 12983-12989 [1989]). The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids.
To provide further insight into the regulation of this important enzyme in DNA synthesis, a need exists for the elucidation of the gene encoding the human dCMP deaminase including the 5'-promoter region.